Plant pathogens secrete effectors to manipulate their host and facilitate colonization. Fusarium oxysporum f. sp. lycopersici is the causal agent of Fusarium wilt disease in tomato. Upon infection, F. oxysporum f. sp. lycopersici secretes numerous small proteins into the xylem sap (Six proteins). Most Six proteins are unique to F. oxysporum, but Six6 is an exception; a homolog is also present in two Colletotrichum spp. SIX6 expression was found to require living host cells and a knockout of SIX6 in F. oxysporum f. sp. lycopersici compromised virulence, classifying it as a genuine effector. Heterologous expression of SIX6 did not affect growth of Agrobacterium tumefaciens in Nicotiana benthamiana leaves or susceptibility of Arabidopsis thaliana toward Verticillium dahliae, Pseudomonas syringae, or F. oxysporum, suggesting a specific function for F. oxysporum f. sp. lycopersici Six6 in the F. oxysporum f. sp. lycopersici- tomato pathosystem. Remarkably, Six6 was found to specifically suppress I-2-mediated cell death (I2CD) upon transient expression in N. benthamiana, whereas it did not compromise the activity of other cell-death-inducing genes. Still, this I2CD suppressing activity of Six6 does not allow the fungus to overcome I-2 resistance in tomato, suggesting that I-2-mediated resistance is independent from cell death.
SummaryPlant-invading microbes betray their presence to a plant by exposure of antigenic molecules such as small, secreted proteins called 'effectors'. In Fusarium oxysporum f. sp. lycopersici (Fol ) we identified a pair of effector gene candidates, AVR2-SIX5, whose expression is controlled by a shared promoter.The pathogenicity of AVR2 and SIX5 Fol knockouts was assessed on susceptible and resistant tomato (Solanum lycopersicum) plants carrying I-2. The I-2 NB-LRR protein confers resistance to Fol races carrying AVR2.Like Avr2, Six5 was found to be required for full virulence on susceptible plants. Unexpectedly, each knockout could breach I-2-mediated disease resistance. So whereas Avr2 is sufficient to induce I-2-mediated cell death, Avr2 and Six5 are both required for resistance. Avr2 and Six5 interact in yeast two-hybrid assays as well as in planta. Six5 and Avr2 accumulate in xylem sap of plants infected with the reciprocal knockouts, showing that lack of I-2 activation is not due to a lack of Avr2 accumulation in the SIX5 mutant.The effector repertoire of a pathogen determines its host specificity and its ability to manipulate plant immunity. Our findings challenge an oversimplified interpretation of the gene-forgene model by showing requirement of two fungal genes for immunity conferred by one resistance gene.
Agroinfiltration is a versatile, rapid and simple technique that is widely used for transient gene expression in plants. In this chapter we focus on its use in molecular plant pathology, and especially for the expression of plant resistance (R) and fungal avirulence (Avr) (effector) genes in leaves of Nicotiana benthamiana. Co-expression of an R gene with the corresponding Avr gene triggers host-defence responses that often culminate in a hypersensitive response (HR). This HR is visible as a necrotic sector in the infiltrated leaf area. Staining of the infiltrated leaves with trypan blue allows visual scoring of the HR. Furthermore, fusion of a fluorescent tag to the recombinant protein facilitates determination of its sub-cellular localization by confocal microscopy. The matching gene pair I-2 and Avr2, respectively from tomato and the fungal root-pathogen Fusarium oxysporum f. sp. lycopersici, is presented as a typical example.
Plant pathogens secrete small proteins, of which some are effectors that promote infection. During colonization of the tomato xylem vessels the fungus Fusarium oxysporum f.sp. lycopersici (Fol) secretes small proteins that are referred to as SIX (Secreted In Xylem) proteins. Of these, Six1 (Avr3), Six3 (Avr2), Six5, and Six6 are required for full virulence, denoting them as effectors. To investigate their activities in the plant, the xylem sap proteome of plants inoculated with Fol wild-type or either AVR2, AVR3, SIX2, SIX5, or SIX6 knockout strains was analyzed with nano-Liquid Chromatography-Mass Spectrometry (nLC-MSMS). Compared to mock-inoculated sap 12 additional plant proteins appeared while 45 proteins were no longer detectable in the xylem sap of Fol-infected plants. Of the 285 proteins found in both uninfected and infected plants the abundance of 258 proteins changed significantly following infection. The xylem sap proteome of plants infected with four Fol effector knockout strains differed significantly from plants infected with wild-type Fol, while that of the SIX2-knockout inoculated plants remained unchanged. Besides an altered abundance of a core set of 24 differentially accumulated proteins (DAPs), each of the four effector knockout strains affected specifically the abundance of a subset of DAPs. Hence, Fol effectors have both unique and shared effects on the composition of the tomato xylem sap proteome.
Increasing numbers of infectious crop diseases that are caused by fungi and oomycetes urge the need to develop alternative strategies for resistance breeding. As an alternative for the use of resistance (R) genes, the application of mutant susceptibility (S) genes has been proposed as a potentially more durable type of resistance. Identification of S genes is hampered by their recessive nature. Here we explore the use of pathogen-derived effectors as molecular probes to identify S genes. Effectors manipulate specific host processes thereby contributing to disease. Effector targets might therefore represent S genes. Indeed, the Pseudomonas syringae effector HopZ2 was found to target MLO2, an Arabidopsis thaliana homologue of the barley S gene Mlo. Unfortunately, most effector targets identified so far are not applicable as S genes due to detrimental effects they have on other traits. However, some effector targets such as Mlo are successfully used, and with the increase in numbers of effector targets being identified, the numbers of S genes that can be used in resistance breeding will rise as well.
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